Vaccines have been utilized to provide a long term protection against a number of disease conditions by very limited administration of a prophylactic agent that stimulates an organism's immune system to destroy disease pathogens before they can proliferate and cause a pathological effect. Various approaches to vaccines and vaccinations are described in Bernard R. Glick and Jack J. Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA, Second Edition, ASM Press pp. 253-276 (1998).
Vaccination is a means of inducing the body's own immune system to seek out and destroy an infecting agent before it causes a pathological response. Typically, vaccines are either live, but attenuated, infectious agents (virus or bacteria) or a killed form of the agent. A vaccine consisting of a live bacteria or virus must be non-pathogenic. Typically, a bacterial or viral culture is attenuated (weakened) by physical or chemical treatment. Although the agent is nonvirulent, it can still elicit an immune response in a subject treated with the vaccine.
An immune response is elicited by antigens, either specific macromolecules, or an infectious agent. These antigens are generally either proteins, polysaccharides, lipids, or glycolipids, which are recognized as “foreign” by lymphocytes known as B cells and T cells. Exposure of both types of lymphocytes to an antigen elicits a rapid cell division and differentiation response, resulting in the formation of clones of the exposed lymphocytes. B cells produce plasma cells, which in turn, produce proteins called antibodies (Ab), which selectively bind to the antigens present on the infectious agent, thus neutralizing or inactivating the pathogen (humoral immunity). In some cases, B cell response requires the assistance of CD4+ helper T cells.
The specialized T cell clone that forms in response to the antigen exposure is a cytotoxic T lymphocyte (CTL), which is capable of binding to and eliminating pathogens and tissues that present the antigen (cell-mediated or cellular immunity). In some cases, an antigen presenting cell (APC) such as a dendritic cell, will envelop a pathogen or other foreign cell by endocytosis. The APC then processes the antigens from the cells, and presents these antigens in the form of a histocompatability molecule:peptide complex to the T cell receptor (TCR) on CTLs, thus stimulating an immune response.
Humoral immunity characterized by the formation of specific antibodies is generally most effective against acute bacterial infections and repeat infections from viruses, whereas cell-mediated immunity is most effective against viral infection, chronic intracellular bacterial infection, and fungal infection. Cellular immunity is also known to protect against cancers and is responsible for rejection of organ transplants.
Antibodies to antigens from prior infections remain detectable in the blood for very long periods of time, thus affording a means of determining prior exposure to a pathogen. Upon re-exposure to the same pathogen, the immune system effectively prevents reinfection by eliminating the pathogenic agent before it can proliferate and produce a pathogenic response.
The same immune response that would be elicited by a pathogen can also sometimes be produced by a non-pathogenic agent that presents the same antigen as the pathogen. In this manner, the subject can be protected against subsequent exposure to the pathogen without having previously fought off an infection.
Not all infectious agents can be readily cultured and inactivated, as is required for vaccine formation, however. Modern recombinant DNA techniques have allowed the engineering of new vaccines to seek to overcome this limitation. Infectious agents can be created that lack the pathogenic genes, thus allowing a live, nonvirulent form of the organism to be used as a vaccine. It is also possible to engineer a relatively nonpathogenic organism such as E. coli to present the cell surface antigens of a pathogenic carrier. The immune system of a subject treated with such a transformed carrier is “tricked” into forming antibodies to the pathogen. The antigenic proteins of a pathogenic agent can be engineered and expressed in a nonpathogenic species and the antigenic proteins can be isolated and purified to produce a “subunit vaccine.” Subunit vaccines have the advantage of being stable, safe, and chemically well defined; however, their production can be cost prohibitive.
A new approach to vaccines has emerged in recent years, broadly termed genetic immunization. In this approach, a gene encoding an antigen of a pathogenic agent is operably inserted into cells in the subject to be immunized. The treated cells are transformed and produce the antigenic proteins of the pathogen. These in vivo-produced antigens then trigger the desired immune response in the host. The genetic material utilized in such genetic vaccines can be either a DNA or RNA construct. Often the polynucleotide encoding the antigen is introduced in combination with other promoter polynucleotide sequences to enhance insertion, replication, or expression of the gene.
DNA vaccines encoding antigen genes can be introduced into the host cells of the subject by a variety of expression systems. These expression systems include prokaryotic, mammalian, and yeast expression systems. For example, one approach is to utilize a viral vector, such as vaccinia virus incorporating the new genetic material, to innoculate the host cells. Alternatively, the genetic material can be incorporated in a vector or can be delivered directly to the host cells as a “naked” polynucleotide, i.e. simply as purified DNA. In addition, the DNA can be stably transfected into attenuated bacteria such as Salmonella typhimurium. When a patient is orally vaccinated with the transformed Salmonella, the bacteria are transported to Peyer's patches in the gut (i.e., secondary lymphoid tissues), which then stimulate an immune response.
DNA vaccines provide an opportunity to immunize against disease states that are not caused by traditional pathogens, such as genetic diseases and cancer. Typically, in a genetic cancer vaccine, antigens to a specific type of tumor cell must be isolated and then introduced into the vaccine.
One of the major obstacles for achieving a tumor-specific immune response is to overcome peripheral T cell tolerance against tumor self-antigens and induce cytotoxic T lymphocytes (CTLs), which effectively eradicate disseminated tumor metastases and subsequently maintain a long-lasting immunological memory preventing tumor recurrence. Human carcinoembryonic antigen (CEA) is an oncofetal membrane glycoprotein, which provides a relevant tumor self-antigen target for the development of DNA vaccines for immunotherapy. A useful animal model for CEA-based vaccines is reported by Clarke et al. Cancer Res. 1998, 58:1469. The model involves the establishment of a mouse line that carries the genomic DNA transgene for human CEA and expresses CEA in a tissue-specific manner similar to humans. Following in vivo priming with CEA-transfected fibroblasts, anti-CEA CD8+ T cells have been elicited in these transgenic mice, which were tolerant to CEA in the CD4+ T cell compartment, described by Mizobata et al. Cancer Immunol. Immuother. 2000, 49:285. Studies in humans by Tsang et al. J Nat'l Cancer Inst. 1995, 87:982, have indicated that CD8+ CTLs specific for CEA are not negatively selected, similar to findings obtained with transgenic mice.
The biological roles of CD40 ligand (CD40L), particularly its interaction with CD40 expressed on antigen presenting cells during costimulation of T cell activation, are well known in the art. CD40 is a 48 kDa glycoprotein expressed on the surface of all mature B cells, most mature B-cell malignancies, and some early B-cell acute lymphocytic leukemias, but it is not expressed on plasma cells, Clark, Tissue Antigens 1990, 35:33-36. CD40L, a type II membrane protein of 35 kDa and a member of the tumor necrosis factor (TNF) gene family, is expressed on the surface of T cells upon antigen recognition. Members of the TNF family are biologically most active when expressed as homotrimers. CD40L is no exception in this regard and can be expressed as a homotrimer (CD40LT) by modification of a 33 amino acid leucine zipper motif fused to the N-terminus of the entire extracellular domain of this ligand. CD40LT DNA has been reported by Gurunathan et al. J. Immunol. 1998, 161:4563, to enhance cellular immune responses such as induction of IFN-γ and cytolytic T cell activity when mice were vaccinated with DNA encoding the highly immunogenic model antigen β-galactosidase.
CD40L is critically involved in the activation of T cells necessary to induce an effective protective immunity against tumor self-antigens. Once MHC class I antigen:peptide complexes are taken up by dendritic cells (DCs) and presented to naive T cells, the first antigen signal is delivered via T cell receptors (TCR), followed by upregulation of CD40L. On the T cell surface, CD40L then induces costimulatory activity on DCs via CD40-CD40L interactions. Thus primed, these APCs now express costimulatory molecules B7.1 (CD80) and B7.2 (CD86), which send a second costimulatory signal to T cells via interaction with CD28, an event required for full activation of T cells to concurrently produce pro-inflammatory cytokines INF-γ and IL12, and to perform effector functions.
An effective means of enhancing efficacy of DNA vaccines is to grow the plasmid encoding DNA in a non-replicating strain of Salmonella typhimurium, which can then be applied as an oral vaccine. The live, attenuated bacteria transport the DNA through the gastrointestinal tract and then through the M cells that cover the Peyer's patches of the gut. From there the attenuated bacteria enter APCs such as dendritic cells and macrophages, where they die, because of their mutation, liberating multiple copies of the DNA inside the phagocytes.
Attenuated bacteria are believed to provide a “danger signal” and stimulate the innate immune system, producing pro-inflammatory cytokines like IL12 and mediators such as nitric oxide that enhance antigen presentation and promote TH1-type cellular immune responses associated with the eradication of tumors. In fact, attenuated S. typhimurium has been reported to be an effective carrier for an autologous oral DNA vaccine that protects against murine melanoma (Xiang et al. Proc. Nat Acad. Sci (USA) 2000, 97:5492). A recombinant Listeria monocytogenes vaccine was reported to be highly effective in mediating regression of primary murine melanoma and their established lung metastases (Pan et al. Cancer Res. 1999, 59:5254). L. monocytogenes produces a strong cellular immune response since, unlike most other intracellular bacteria, it escapes into the cytoplasm by disrupting the phagosomal membrane thus allowing any protein it secretes to target both MHC class I and class II pathways of the infected cell for antigen presentation.
Xiang et al., Clin. Cancer Res., 2001, 3:8565, reports on partial tumor-protection against a lethal challenge of MC38 murine colon carcinoma cells, stably transduced with CEA and KSA, a human pan-epithelial cell adhesion molecule. Mice were vaccinated by oral gavage with a CEA-based DNA vaccine carried by attenuated Salmonella typhimurium, which induced MHC class I antigen-restricted CD8+ T cell responses, resulting in rejection of subcutaneous tumors. However, this occurred in only some of the experimental mice transgenic for CEA, even when boosted with a recombinant antibody-IL2 fusion protein that targeted IL2 to the tumor microenvironment.
There is an ongoing need for vaccines that elicit a CD8+ T cell-mediated tumor-protective immune response against CEA self-antigen with improved efficacy against colon cancer. The present invention accomplishes this goal with a unique, dual function DNA vaccine encoding CEA and CD40LT, activating both DCs and naive T lymphocytes, particularly when aided by boosts with huKS1/4-IL2 fusion protein.